PROJECT SUMMARY. TP53 is the most frequently mutated tumor suppressor gene in human cancer. However, about half of cancers retain a wild type version of the p53 protein, which is commonly attenuated by other mechanisms, such as overexpression of the repressor MDM2. The presence of wild type p53 opens a window of opportunity for therapeutic reactivation of this transcription factor. The goal of this proposal is to elucidate the molecular mechanisms that modify the p53 network in a cell type-specific manner, affecting cell fate choice and tumor suppression downstream of p53 reactivation. The ultimate goal is to enable effective p53-based targeted therapies. Using a comprehensive multi-omics experimental and analytical pipeline we identified three major regulatory mechanisms affecting p53 signaling in a cell type-specific fashion: chromatin architecture at target loci, secondary ?amplifying? transcription factors, and cell type-specific translational regulators. We will investigate each mechanism in detail by developing the following Specific Aims: 1. To define the impact of cellular transformation on the direct p53 program. We discovered that cell type-specific variations in the direct p53 transcriptional program are associated with distinct patterns of chromatin architecture, including differences in DNA methylation. We will now test the hypothesis that the direct p53 program is attenuated during tumor evolution via chromatin-based mechanisms that restrict the number of available direct p53 targets. We will do this by applying our multi-omics pipeline to study the direct p53 program in intestinal organoids derived from normal epithelium and colorectal carcinoma explants. 2. To define the role of secondary transcription factors in the p53 response. We identified two novel direct p53 target genes, the transcription factors ETV7 and POU2F2, which are predicted to elicit the indirect transcriptional program in cells where p53 has an intact tumor suppressive activity. We will define the role of ETV7 and POU2F2 in the observed amplification of p53 effects on the global transcriptome, as well as their contribution to p53-dependent cellular responses in vitro, and tumor suppressive activity in xenograft models. 3. To identify translational regulators controlling the cellular response to p53 activation. Translation of hundreds of mRNAs is activated or repressed downstream of p53 in a cell type-specific manner. We identified 3?UTR motifs and RNA-binding proteins (RBPs) that are predicted to mediate cell type-specific translational control. We will continue to identify and characterize functional RNA motifs and interacting RBPs acting in different contexts, and define their contribution to p53-dependent cellular responses in vitro and to the ability of p53 to induce tumor regression in xenograft models. Upon completion, this proposal will significantly advance our understanding of regulatory mechanisms within the p53 network, and contribute to the future design of improved p53-based therapeutic strategies.